In accordance with recent development of genetic engineering and biology, there are many attempts to produce or obtain a large amount of specific protein to be used for treatment of various types of industries and diseases. Accordingly, protein combination technology, mass-production technology, and purification technology, and the like, for obtaining a desired protein have been intensively developed.
Frequently, the target protein to be required by human may be produced by culturing a cell transformed with a vector expressing the target protein so that the target protein is expressed. Occasionally, the protein may be expressed in eukaryotic cells, prokaryotic cells, and the like, and in specific cases, the protein may be expressed in transformed plants or transformed animals. For example, a method of expressing a protein in transformed animals that secrets milk to obtain the target protein through the milk of the transformed animals, and the like, has been attempted. In this case, the target protein may be isolated and refined through cell culture or milk.
In a case of expressing a protein in animals and plants or microorganisms which methods for obtaining a target protein through separate secretion do not exist, processes for extracting a protein from storage organ or an inner part of cells are primarily needed. A process for obtaining the target protein from the transformed cell is not easily performed. Accordingly, a method for recombining a target protein to include a tag rather than a wild-type one has been largely used to easily obtain the protein.
A method using a tag for purification is one of methods in which significantly high efficiency is exhibited among various protein purification technologies, wherein the tag to be used is largely classified into a peptide tag and a protein tag. The peptide tag consists of short amino acids and includes a his-tag (histidine-tag) as a representative one. Particularly, a hexahistidine tag (His6-tag) has been largely used. Histidine peptide has specific chemical affinity to nickel, such that fusion proteins including corresponding tags are possible to be refined with high purity by column including nickel. The protein tag is a tag including corresponding domains, and the like, in order to use characteristics, and the like, of domains of proteins bound to specific components. The protein tag includes a GST-tag (Glutathione S-transferase-tag). The GST tag may be refined with high purity by column using glutathione which is a substrate of GST as a fixing media.
The tag fused and expressed in the target protein for protein purification as described above may have a risk of interrupting structure or function of the target protein itself, such that a method for obtaining the target protein from which the tag is cleaved has been considered. Meanwhile, the conventional method requires a primary process for obtaining a protein including a tag, a process for cleaving the tag, and a process for purifying a target protein only. During these processes, the target protein is lost, an amount of finally obtained protein is decreased, and cost and time for corresponding processes are also excessive. Accordingly, it is required to develop a method for minimizing the loss of the target protein in the process for cleaving the tag, and purifying the protein rapidly, while maintaining advantages of the method for purifying a protein using the tag.
Under this background, a method for purifying a protein using domain of Sortase A having cleaving function protein having self-cleaving function and cleavage site sequence recognized by the corresponding domain was developed (Mao H et al., Protein Expr. Purif. 2004; 37(1):253-63). The Sortase A (SrtA, 60-206 A.A.) is an enzyme which recognizes the cleavage site sequence (LPXTG, X is an any amino acid) in circumstance in which there are calcium and triglycine to generate a catalytic reaction which cuts between threonine (T) and glycine (G). The method for purifying a protein using the conventional Sortase A is a method including a step of producing a recombinant expression vector including polynucleotide encoding a tag-Sortase A(60˜206 A. A.)-LPXTG-a target protein, expressing the protein in a host cell, and binding host cell pulverized product to a tag binding column; a step of removing impurities; a step of injecting calcium and/or triglycine-containing solution and performing a reaction; and a step of obtaining the protein to be capable of purifying the protein and removing a tag at a time with the use of the column only once. However, the method of using the conventional domain in Sortase A having cleaving function has a problem in that purification efficiency is low, according to a target protein.
Therefore, the present inventors has completed the present invention by confirming that remarkable protein yield is possibly obtained by focusing on a direction of binding the domain in Sortase A having cleaving function in a fusion protein and applying a linker between the Sortase A and site of sequence for cleavage, as compared the conventional method.